| Noncovalent chemistry is the basis of many important biological interactions, including enzyme-ligand complex formation. As these interactions are typically weaker than covalent bonds, special analysis methods are needed for studying noncovalent complexes. Using gentle ionization methods like electrospray ionization (ESI), noncovalent complexes can be preserved and analyzed by mass spectrometry. Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) provides superior mass accuracy, resolving power, and tandem-in-time capabilities. Among the dissociation techniques available are in-source collision-induced dissociation and infrared multiple photon dissociation (IRMPD).;Dynamic combinatorial chemistry uses a special type of library wherein reversible binding between library members and between library members and a target molecule expands the potential number of strong complex interactions. Here, several 2', 3'-cyclic monophosphate nucleotides were incubated with Ribonuclease A to generate enzyme-ligand complexes. Enzymatic activity may drive the library members to form RNA chains via phosphodiester bond generation.;This dissertation shows the method development for screening a cyclic nucleotide-based dynamic combinatorial library for tight-binding ligands of Ribonuclease A using ESI-FTICR-MS. A model system comprised of Ribonuclease A and a known inhibitor (uridine 5' monophosphate) was used to optimize mass spectrometry parameters. The resultant noncovalent enzyme-inhibitor and inhibitor-inhibitor complexes were studied by IRMPD. Tandem MS was demonstrated for complex dissociation and inhibitor fragmentation in a single experiment.;Next, the use of variable wavelength dissociation of noncovalent complexes was employed to optimize dissociation efficiency. Specifically-bound Ribonuclease A-Library complexes were compared to nonspecific control complexes. Differences were observed at two of the wavelengths employed, with specific complexes requiring greater energy for complete dissociation than nonspecific complexes.;Lastly, two small dynamic combinatorial libraries were screened for binding affinity to Ribonuclease A, and certain of the binding species identified. A microdialysis method was developed to reduce signal suppression due to the high library concentrations required for proper library functioning. The screening method efficacy was limited by exact mass redundancy between the species in the libraries. Additional dissociation steps were unsuccessful at generating sufficient signal to discriminate between the structures with identical masses. Future work should be devoted to expanding library sizes and developing methods to use higher library concentrations. |
